Background: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine that activates apoptosis through death receptors on the cell surface and is regarded as a potential anticancer agent. However, many cancer cells are resistant to TRAIL-induced apoptosis. Objective: The aim is to identify the herbal medicines that could help overcome resistance in TRAIL-resistant lung cancer cells. Materials and Methods; TRAIL-resistant A549 cells and 13 herbal medicines with known apoptosis-related anticancer effects were used in this study: Clematidis Radix, Corydalis Tuber Rhizoma, Paeoniae Radix Rubra, Corni Fructus, Curcumae longae Rhizoma (CLR), Moutan Cortex, Salviae miltiorrhizae Radix, Phellodendri Cortex, Farfarae Flos, Paeoniae Radix Alba, Angelicae gigantis Radix, Coptidis Rhizoma (CR), and Taraxaci Herba. Cytotoxic effects were investigated after a 48-h incubation, using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, to identify the herbal medicines with the most potent synergistic effects with TRAIL. Results: The majority of the 13 medicines exhibited concentration-dependent cytotoxicity against A549 cells. Among them, CR and CLR showed the most potent cytotoxic effects, based on the IC50. We then investigated the use of these two medicines in combination with TRAIL and identified synergistic cytotoxic effects against TRAIL-resistant A549 cells. Conclusion: Synergistic cytotoxic effects of the combination of TRAIL and herbal medicines, in particular, CR and CLR, were confirmed in A549 cells. Therefore, CR and CLR showed potential to be used as candidates to overcome TRAIL resistance. Future studies to identify their underlying mechanism of action are required.Abbreviations used: TRAIL: Tumor necrosis factor-related apoptosis-inducing ligand; CLR: Curcumae longae Rhizoma; CR: Coptidis Rhizoma; NSCLC: non-small cell lung cancer.

Apoptosis, a genetically programmed and active cell suicide process, plays a crucial role in homeostasis and eliminating unwanted cells. Evasion of programmed cell death is one of the important genetic changes in malignant transformation. Therefore, apoptosis induction is a potential therapeutic goal of cancer treatment.[1]

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has long been regarded as an effective target for cancer therapy. It is a small molecule, known to induce apoptosis selectively in cancer cells through interaction with the death receptors DR4 and DR5, while not damaging normal cells.[2] There have been several single-agent clinical trials, including phase 1 and 2 trials, evaluating the efficacy of various TRAIL receptor agonists. However, because of resistance, their anticancer effects have proven to be insufficient.[3] Therefore, much effort has focused on overcoming TRAIL resistance in cancer cells through the use of various natural products.[4],[5]

In traditional Korean medicine, herbal medicines have been used in the treatment of many diseases, including cancer, for centuries, because of their safety, efficacy, and inexpensiveness. The efficacy of a number of herbal medicines in the treatment of lung cancer has been well documented.[6] Thus, for the present study, we obtained water extracts of herbal medicines with demonstrated apoptosis-related antitumor activities and screened them based on their anti-proliferative effects. We then evaluated their TRAIL-sensitizing effects in A549 TRAIL-resistant lung cancer cells.

To evaluate the cytotoxic effect of the herbal extracts, cell viability was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma Chemical Co., St. Louis, MO) assay. Briefly, the cells were seeded in 96-well plates at a density of 1 × 104 cells per well and incubated for 48 h. They were then treated with various concentration of herbal extracts (0, 0.01, 0.1, 10, 100, 1000, and 10000 μg/ml). After incubation for a further 48 h, 200 ml of MTT (0.5 mg/ml) was added to each well. Followed by 4-h incubation, absorbance was measured using an ELISA reader. Absorbance was measured using an ELISA reader (Molecular Devices, Sunnyvale, CA, USA) at 540 nm. MTT assays were carried out in triplicate. The IC50 was defined as the concentration of herbal medicine water extract that inhibited cell growth by 50% relative to the untreated control.

Cell viability was again investigated using MTT assay to evaluate the synergistic effects of combinations of herbal extracts and TRAIL on the proliferation of A549 cells. The cells were seeded at a density of 1 × 104 cells per well and incubated for 48 h and were then treated with 0, 100, 200, or 400 μg/ml of herbal extracts and 40 ng/ml of TRAIL concurrently. MTT assays were then used to assess cell viability.

Combination index calculation

The combination index (CI) for the treatment combining herbal extracts (CLR and CR) and TRAIL was calculated using CompuSyn software and Chou–Talalay median-effect analyses.[7] The efficacy of each drug combination was represented by its CI value, with CI <1 indicating synergism, CI = 1 indicating an additive effect, and CI >1 indicating antagonism.

Results

Cytotoxic activities of aqueous herbal extracts on A549 cells

To investigate the cytotoxic activity of whole-plant water extracts, A549 cells were incubated with different herbal water extract concentrations for 48 h. Each herbal water extract exhibited some degree of inhibition on the proliferation of A549 cell in a concentration-dependent manner and IC50 values were calculated [Table 1]. The water extracts of CR and CLR were determined to have the most potent inhibitory effects. Therefore, we selected CR and CLR as candidate drugs for maximum synergy with TRAIL against A549 cells.

Resistance to TRAIL was confirmed by evaluating its cytotoxicity against A549 cells at different concentrations [Figure 1]a. In addition, to identify synergistic effects of TRAIL and aqueous herbal extracts, the cytotoxicity of CR and CLR at different doses were again investigated, and their cytotoxicity in combination with a fixed concentration of 40 ng/ml TRAIL, which had been confirmed to have no cytotoxic effect on A549 cells, were determined. The observed cytotoxicity for each single and combination therapy was then compared. The results of this analysis indicated that single-agent treatment inhibited the proliferation of A549 cells in a dose-dependent manner, while combination treatment further inhibited proliferation at several concentrations in a synergistic manner [Figure 1]b and [Figure 1]c.

Combination effects were assessed using the Chou–Talalay method, which is based on the median effect equation. From the affected fraction (Fa)–CI plot, the efficacy of each combination was determined. Both CR and CLR combined with TRAIL had CI values <1, suggesting synergism against A549 cell lines, and implying the overcoming of resistance to TRAIL [Figure 1]d and [Figure 1]e.

Discussion

Lung cancer is a major cause of cancer-related deaths worldwide and is divided into small cell and nonsmall cell lung cancers (NSCLC). The latter are less sensitive to conventional chemotherapeutic agents. Therefore, the development of novel therapeutic strategies, including induction of apoptosis, is necessary.[1] TRAIL is regarded as an attractive therapeutic agent because it directly induces apoptosis that selectively eliminates tumor cells without harming normal cells.[2] However, TRAIL resistance has been observed in several types of NSCLC, and clinical trials using TRAIL therapies have shown disappointingly weak therapeutic effects, especially when TRAIL agonists are used as a monotherapy.[1] Thus, it is important to find novel drugs that act synergistically with TRAIL and can also overcome this resistance.[3]

There have been some studies on overcoming TRAIL resistance in various cancers, including lung cancer, using herbal medicines, and natural compounds.[4],[8],[9] To the best of our knowledge, none of the studies has examined TRAIL-resistant NSCLC. Therefore, in this study, we investigated the antitumor effects of 13 frequently used herbal medicines containing compounds associated with or related to apoptosis and assessed their ability to overcome TRAIL resistance in A549 cells.

It is well documented that CR and CLR have antitumor effects against various cancers.[10] Specifically, berberine, from CR, has been shown to enhance TRAIL-induced apoptosis in breast cancer.[11] In addition, curcumin, from CLR, is known to have excellent antitumor effects and to reduce TRAIL resistance in prostate cancer.[12] However, clinically, herbal medicines are most commonly used as decocted aqueous extracts,[13] and to date, no study has investigated the effects of total-herb aqueous extracts of CR and CLR on overcoming TRAIL resistance in NSCLC. In this study, CR and CLR aqueous extracts have been shown to not only act synergistically with TRAIL but also to overcome TRAIL resistance. Animal experiments and studies on the molecular mechanisms of action of these extracts should be conducted in the near future to establish a foundation for clinical trials.

Conclusion

In the present study, we confirmed CR and CLR to be the most potent of the 13 anticancer herbal medicines known to induce apoptosis. We further investigated whether these two medicines could reduce resistance to TRAIL, an anticancer cytokine causing apoptosis, and examined the synergistic effect of combinations of TRAIL and these herbal medicines. We found that combination treatment with TRAIL and CR or CLR resulted in greater cytotoxicity than single-agent treatment in A549 TRAIL-resistant cells, indicating that TRAIL resistance was reduced by combination therapy.

Acknowledgements

This work was supported by a grant from Kyung Hee University in 2015.(KHU-20150848).